The rate at which erythrocytes or red blood cells settle through blood plasma in a whole blood specimen has long been the subject of medical study. It has been found that the sedimentation rate of blood can be significantly increased by a wide range of inflammatory conditions and diseases. Various attempts have been made to automate blood sedimentation apparatus and to correlate settling or sedimentation rates and patterns to inflammatory conditions.
The original laboratory studies, however, are still regarded as the standards. More particularly, there is a Wintrobe sedimentation method and a Westergren sedimentation method. The Westergren method is most widely used and employs a 300 millimeter long settling tube with the lower 200 millimeters being graduated. The tube is filled to the 200 millimeter mark with approximately 0.8 milliliter of blood and 0.2 milliliters of anticoagulant diluent which is allowed to gravity settle over a one or a two hour period. The amount of settling in a one hour period in a Westergren settling tube is generally regarded as the standard for blood sedimentation rate.
In any blood sedimentation study the specimen is first thoroughly mixed so that the erythrocytes are evenly distributed throughout the specimen. In the Westergren method, after mixing, the 300 millimeter tube is brought to a vertical orientation for gravity settling and the settling clock started. After one hour the amount of settling which has occurred, as determined by the distance that the interface between the plasma and the erythrocytes has traveled downward, is measured.
Various attempts have been made to automate the Westergren settling process. U.S. Pat. No. 4,041,502 to Williams, et al., for example, discloses an automated sedimentation measuring system in which measurements are taken every 15 seconds for one hour (or two hours) and a blood sedimentation curve is produced as a result of these measurements. U.S. Pat. No. 4,848,900 to Kuo, et al. is a similar blood sedimentation automated system in which a blood sedimentation curve is also generated over a one hour period.
While the amount of sedimentation varies for each specimen, as influenced by inflammatory conditions, the general shape of blood sedimentation curves is quite similar as a result of the settling phenomena which are operative. Most sedimentation curves, therefore, have three phases which can be clearly identified. First, there is a "lag phase" in which settling is very slow and gradual. Next comes a "decantation phase" in which rapid, virtually linear, settling occurs. Finally, there is a "syneresis phase" in which the rate of settling greatly slows towards the end of the one hour settling period.
While the erythrocytes are more dense than the plasma, they are small and thus have such a high surface area relative to volume that they do not readily sediment through plasma as single cells. In the initial or lag phase, therefore, the erythrocytes must come in contact with each other to group together in clumps or clusters known as "rouleaux." Once a sufficient number of erythrocytes have grouped in rouleaux, the rouleaux will begin to sediment through the blood plasma toward the bottom of the sedimentation tube. Thus, the initial sedimentation or lag phase may take 5 to 15 minutes for sufficient rouleaux clusters to form to enter the more rapid decantation phase. The lag phase portion of a sedimentation vs. time curve, therefore, is relatively flat and typically shows little sedimentation or movement of the plasma-erythrocyte separation interface.
As the erythrocytes in rouleaux settle, they contact other erythrocytes which adhere and decrease the surface are to volume ratio and hence the drag on the sedimenting rouleaux. Additionally, however, the plasma at the bottom of the sedimentation tube must rise or be displaced upwardly by a volume equal to the sedimenting erythrocytes. The settling process, therefore, involves both downward migration or sedimentation of the more dense erythrocytes in rouleaux and upward migration of the lighter plasma through the downwardly migrating rouleaux. The sedimentation rate increases significantly and is fairly linear in the decantation phase or the mid-range of the sedimentation process.
Toward the end of the sedimentation process, however, the erythrocytes begin to pack more tightly at the bottom of the tube. This narrows the pathways for plasma to upwardly migrate to the plasma-erythrocyte separation boundary or interface. The plasma, therefore, has a more difficult time escaping from between the red cells in rouleaux as the packing density or hematocrit rises. Sedimentation, therefore, again slows in this last or syneresis phase of sedimentation.
Various attempts have been made to devise methods and apparatus for accelerating the determination of erythrocyte sedimentation rates. In my U.S. Pat. No. 3,824,841, for example, a sedimentation method is disclosed in which specimens are centrifuged in vertically oriented settling tubes, with the tubes periodically rotated about their longitudinal axis. The erythrocytes seesaw back and forth across the tube and downwardly under a combination of gravity and centrifugal forces. The sedimentation time using this process is reduced from one hour to about three minutes, and the sedimentation rates measured using this process and apparatus can be related to the Westergren method using non-linear regression algorithms. This apparatus and method, however, have drawbacks in the form of the complexity of the apparatus, as well as the need to use non-linear regression algorithms.
Sedimentation studies also have been undertaken in shorter tubes and particularly 100 millimeter settling tubes. The problem with this technique is that the final or syneresis phase, in which the hematocrit is rapidly rising, occurs earlier, again requiring non-linear algorithms for correlation to Westergren sedimentation results.
Additionally, accelerated blood sedimentation has been measured using tilted or inclined sedimentation tubes instead of vertically oriented tubes. Using an inclined tube (a tube 100-200 millimeters long and 2.5 millimeters in diameter inclined at about 30 or 45 degrees from vertical) settling rates can be measured after only 20 minutes of settling. The settling rate which is determined using such apparatus, however, is not a true Westergren sedimentation rate because once again the relationship between the hematocrit rise and the entrapment of plasma is altered resulting in a nonlinear relationship between the test method. Thus, the settling is non-linear and the measured rates must be related to Westergren rates by non-linear correlation algorithms. Tilting of the tube reduces the settling time by about two-thirds but because there is in this method no shortening of the lag phase, 20 minutes is still required and a non-linear correlation to Westergren rates is also necessary.
In general, the use of non-linear algorithms becomes less reliable in relating results to Westergren sedimentation rates as the sedimentation rate increases. High sedimentation rates usually indicate the presence of inflammatory conditions. Thus, the non-linear effects induced by rapid sedimentation tend to decrease the correlation accuracy to Westergren rates for specimens which are most affected by disease and other conditions sought to be discovered or analyzed by the sedimentation process.
Mixing of small volumes of liquid specimens has been found to be very difficult. One approach has been to provide a specimen container which has a septum or wall extending down most of the length of the tube except the ends. Such a structure is shown in U.S. Pat. No. 4,701,305 to Hattori et al. The Hattori et al. approach, however, requires that the specimen be urged in a loop around the septum with the result that mixing of the specimen constituents is relatively slow.
Accordingly, it is an object of the present invention to provide an apparatus and method for erythrocyte blood sedimentation which can be rapidly accomplished and yet is capable of high correlation by linear transposition to Westergren sedimentation rates.
Another object of the present invention is to provide an erythrocyte sedimentation method and apparatus which can be used repeatedly on the same specimen to rapidly determine and verify the blood sedimentation rate.
Another object of the present invention is to provide a blood sedimentation apparatus and method in which the whole blood specimen, from the moment of venipuncture, remains in a sealed container.
Still a further object of the present invention is to provide a blood sedimentation apparatus and method in which smaller blood specimens are required and mixing of the blood specimen can be readily accomplished in very small volumes.
Another object of the present invention is to provide an improved specimen container for use with processes requiring mixing of small liquid volumes.
Still another object of the blood sedimentation apparatus and method of the present invention is to provide a sedimentation process having increased accuracy, relatively low cost, and suitability for semi-automated use by relatively unskilled paramedical personnel.
The blood sedimentation method and apparatus of the present invention have other objects and features of advantage which will become apparent from, and are set forth in more detail in, the accompanying drawing and following Best Mode Of Carrying Out The Invention.